Association of Entamoeba gingivalis with Periodontal Disease—Systematic Review and Meta-Analysis

The oral cavity is a habitat to a diverse range of organisms that make up an essential element of the human microbiota. There are up to 1000 species of micro-organisms capable of colonizing the mouth. Thirty percent of them are uncultivable. The genus Entamoeba includes several species, out of which at least seven of them are able to inhabit the human body (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba coli, Entamoeba polecki, Entamoeba hartmann, Entamoeba gingivalis). It was shown that only E. gingivalis is able to colonize the oral cavity. The aim of this study was to evaluate the association and prevalence of E. gingivalis in periodontal disease using two electronic database search engines. In order to have a broader view of the subject, a comprehensive manual search was conducted between 15th February 2023 and 1 April 2023 on these content aggregators and the initial search resulted in 277 articles using the keywords “E. gingivalis”, “periodontitis”, “E. gingivalis”, “periodontal disease”, “prevalence”, and “incidence”, in different combinations. The results showed that 755 patients were infected with E. gingivalis out of a total number of 1729 patients diagnosed with periodontal disease, indicating a global prevalence of 43% in the set of patients analyzed. E. gingivalis was prevalent in 58% of the patients that had gingivitis and in 44% of the patients with periodontitis. Prevalence of E. gingivalis based on gender was 43% in female patients and 47% in male patients. The results indicate that the higher incidence of E. gingivalis in people with periodontal disease compared to healthy people is more than just a sign of the disease; it could also be linked to the severity of the condition and the disease propensity to progress.


Introduction
Due to the aging population's growth and higher preservation of natural teeth, it is anticipated that the prevalence of periodontal diseases, namely gingivitis and periodontitis, will rise globally in the years to come [1].The Center for Disease Control and Prevention (CDC) showed in 2021 that the prevalence of periodontitis in the United States was 47.2% [2], while Holtfreter et al. [3] showed a prevalence of 70% in German adult patients [3].Periodontitis is a disease in which the prevalence increases with age.Therefore, to understand and treat the disease better, it is substantial to know the causing factors.The major factors are bad oral hygiene, smoking, and altered general status like diabetes and hereditary factors.The composition of the oral microbiota also plays a substantial role in the onset of the disease [4,5].The oral cavity is home to a diverse range of organisms that make up an essential element of the human microbiota.There are up to 1000 species of organisms capable of colonizing the mouth [6].Some of these micro-organisms have the ability to destroy certain structures in the mouth [6].The usual approach is to destroy tissues in the oral cavity as in periodontal diseases where the microbiota destroys the periodontium.
While gingivitis usually refers to the earliest form of gum disease, periodontitis is an inflammatory disease, which, unlike gingivitis, is a progressive disease that is characterized by the destruction of the periodontium tissues and clinical attachment loss [4][5][6][7].The most characteristic clinical signs of periodontitis, such as bleeding on probing, development of periodontal pockets and gingival recessions, furcation involvement, and presence of radiographic bone loss are frequently observed in these patients [5][6][7].In patients with healthy immune systems, periodontium can cope with the presence of bacteria through a variety of immune system activities.When the infection control system is disrupted by subgingival bacteria such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tanerella forsycia, and Treponema denticola the adaptive response is triggered [2,6].The severity of the disease is partly determined by the host's reaction to this inflammation that is caused by the micro-organisms, the immunologic defense, and the composition of the oral microbiota [8].Bacteria that are capable of inducing the immunologic defense and the inflammatory reaction are part of the so-called "red complex".The presence of these species in periodontitis was confirmed in recent studies [9,10].Since the etiology of periodontitis is still unclear and it is classified by the WHO as a non-communicable disease, it is crucial to identify its pathophysiology for better knowledge.Due to the fact that Entamoeba species can be correlated with periodontitis, it is important to establish if these species are associated with the occurrence of periodontal disease.Recently, Bao et al. (2020) in their study showed that E. gingivalis can damage periodontal tissues [11].Parasites of this type are unicellular organisms and include at least seven species (E.histolytica, E. dispar, E. moshkovskii, E. coli, E. polecki, E. hartmanni, E. gingivalis) [12].It was shown that only E. gingivalis is able to colonize the oral cavity [13].E. gingivalis pathogenicity was unclear until Bao et al. [11] showed its implication in periodontal diseases and demonstrated that the prevalence of these species was significantly increased in periodontal pockets of periodontitis.The prevalence in patients with periodontitis and in individuals with periodontal health was 74-88.9%[11,13].Further studies, like Mielnik-Błaszczak et al. [14], showed an increased abundance in other dental conditions like dental caries [14].E. gingivalis was long believed to be an opportunist species with no negative side effect until its effect on the periodontium was proven to be inducing periodontal disease [11].Therefore, the aim of the study was to evaluate the prevalence of these parasite species and its association with periodontal parameters.The current evidence regarding the epidemiology of these species in periodontitis and other periodontal conditions was also investigated.

Materials and Methods
In this literature review, the authors assessed studies that ascertained whether E. gingivalis had any discernible impact on the development of any type of periodontal disease as well as the effects that this species might have on the progression of periodontal disease.Understanding the effect of E. gingivalis on the periodontal structures was the main goal of this study.

Inclusion and Exclusion Criteria
Articles that featured material relevant to the review's objectives and that covered all age groups were chosen for full-text screening.The main manual search was performed up to 1 April 2023, using the Mesh keywords "E.gingivalis", "periodontitis", "gingivitis", "periodontal disease", and "Entamoeba species" in different combinations.The authors included articles that presented randomized/non-randomized investigations, clinical cases with large sample sizes, in-depth case reports, and validated comparative analyses.The year of the publication was not a criterion in the selection process.
Studies involving animal subjects, seminar presentations, academic publications, opinion pieces, and incomplete data were not included in the scope of the examination.
The authors took into account all publications that had been released in relation to our topic and the number of papers was found to be quite sparse.All articles that were written in languages other than English were also disregarded (Table 1).

Data Selection Protocol
Using specific keywords such as "E.gingivalis" "Periodontal Disease" "Periodontitis" "Gingivitis", and "Entamoeba species", two separate reviewers manually combed through relevant publications in Google Scholar and the Pubmed Central database and internet search engines.The selected articles were compared and a third reviewer was consulted in case of disagreement.

Study Selection
A thorough search conducted between 15 February 2023 and 1 April 2023 of the online journals turned up a total of 277 documents.After eliminating 145 articles that were duplicates of each other, only 132 original papers were selected.A further 69 articles were excluded after the abstracts and titles of the submissions were examined.After further selection, based on the essential inclusion and exclusion criteria, 17 documents were ultimately selected.They were mostly clinical cases, in vivo experiments, and comparative analyses (Figure 1).
The authors took into account all publications that had been released in relation to our topic and the number of papers was found to be quite sparse.All articles that were written in languages other than English were also disregarded (Table 1).

Randomized investigations Animal subjects
Non-randomized investigations Seminar/academic publications In-depth case reports

Validated comparative analyses
Incomplete data/opinion pieces Language other than English

Data Selection Protocol
Using specific keywords such as "E gingivalis" "Periodontal Disease" "Periodontitis" "Gingivitis", and "Entamoeba species", two separate reviewers manually combed through relevant publications in Google Scholar and the Pubmed Central database and internet search engines.The selected articles were compared and a third reviewer was consulted in case of disagreement.

Study Selection
A thorough search conducted between 15 February 2023 and 1 April 2023 of the online journals turned up a total of 277 documents.After eliminating 145 articles that were duplicates of each other, only 132 original papers were selected.A further 69 articles were excluded after the abstracts and titles of the submissions were examined.After further selection, based on the essential inclusion and exclusion criteria, 17 documents were ultimately selected.They were mostly clinical cases, in vivo experiments, and comparative analyses (Figure 1).The same two reviewers separately extracted the following information from the selected articles: authors, year of publication, type of publication, study topic, population demographics (n, age), outcome measure(s), pertinent result(s), and conclusion(s), so that the information could be compared latterly.A third reviewer was consulted to go through any discrepancy during data comparison.For quantification of the search results, the PRISMA flow chart guidelines [15] were used.

Quality, Risk of Bias, and Heterogenity Assessment
In order to evaluate the methodological quality of included studies, the articles' data were independently evaluated by the authors using a special manual form designed according to the following categories: study model design, number of subjects, and study results.The quality assessment of the included studies was performed using the Revman Cochrane™ approach.The risk of bias tool identified the domains specified in the Cochrane risk of bias instruments for systematic reviews.All the authors included in the present study clearly defined both their study objective and the population (number, characteristics, and eligibility) on which they were going to carry out the research.Heterogeneity was determined by also using the Cochrane's Q test and I2 index.A fixed effect model would be used for the research if the heterogeneity was p > 0.1 and I2 < 50%, while a random effect model was used if the heterogeneity was p ≤ 0.1 or I2 ≥ 50%.We also determined the 95% Confidence Interval (CI) and Mean Difference (MD) for the remaining continuous and categorical outcomes in each comparison.

Statistical Analysis
Data were selected based on information on the size of samples, variables analyzed, and various other aspects of the research.The data were then loaded into the GraphPad™ software (Dotmatics, Boston, MA, USA) version 6 for Microsoft Windows™.Our analysis generated forest plots showing the odds ratio, risk ratio, and risk difference (using a fixed effects model) of the different clinical results.These plots assume a 95% confidence interval and are rendered in the following figures.

Results
This paper includes clinical trials that monitor the prevalence of E. gingivalis in periodontal disease and compares it to E. gingivalis prevalence in healthy populations, based on gender, age, method, and sample size used.

Results Based on Periodontal Disease
A total of 277 records were recorded through English databases and search engines, PubMed and Google Scholar.The main characteristics of the studies investigated are shown in Table 2.The prevalence of E. gingivalis varied from a low of 17% of cases reported by H. Mahmoudvand et al. [19] to a high of 87%, reported by A. Yaseen [30].The mean prevalence of all studies combined was 43% as shown in Figure 2. Further analysis revealed the prevalence of E. gingivalis in patients with gingivitis was 58% (Figure 3) and 44% (Figure 4) in patients with periodontitis.Seven investigated the E. gingivalis prevalence in gingivitis.The rest of the 11 studies investigated the prevalence of it in periodontitis (Figure 4).

12
Al-Jubory et al. [ Yaseen et al. [30] 2021 143 125 The prevalence of E. gingivalis varied from a low of 17% of cases reported by H. Mahmoudvand et al. [19] to a high of 87%, reported by A. Yaseen [30].The mean prevalence of all studies combined was 43% as shown in Figure 2. Further analysis revealed the prevalence of E. gingivalis in patients with gingivitis was 58% (Figure 3) and 44% (Figure 4) in patients with periodontitis.Seven investigated the E. gingivalis prevalence in gingivitis.The rest of the 11 studies investigated the prevalence of it in periodontitis (Figure 4).

Results Based on Dental Health
Out of 18 studies investigated, 12 studies reported a case-control group.This group was defined as having a heathy periodontal status, with no gingival inflammation or clinical attachment loss.When comparing the prevalence of E. gingivalis in the study groups that presented periodontal disease (43%) with the control group that presented no signs of periodontitis, the prevalence of E. gingivalis was found to be 25%.J. Luszcak et al. [16], M. EL-Dardiry et al. [17], and G. Garcia et al. [18] reported a lower prevalence in the control group than in the rest of the studies compared with the respective periodontal disease group.In all studies investigated, the prevalence of E. gingivalis in control groups was lower than in the periodontal disease groups (Figure 5).The results showed that in cases with periodontal disease, there is a higher prevalence of E. gingivalis as shown by A.

Results Based on Dental Health
Out of 18 studies investigated, 12 studies reported a case-control group.This group was defined as having a heathy periodontal status, with no gingival inflammation or clinical attachment loss.When comparing the prevalence of E. gingivalis in the study groups that presented periodontal disease (43%) with the control group that presented no signs of periodontitis, the prevalence of E. gingivalis was found to be 25%.J. Luszcak et al. [16], M. EL-Dardiry et al. [17], and G. Garcia et al. [18] reported a lower prevalence in the control group than in the rest of the studies compared with the respective periodontal disease group.In all studies investigated, the prevalence of E. gingivalis in control groups was lower than in the periodontal disease groups (Figure 5).The results showed that in cases with periodontal disease, there is a higher prevalence of E. gingivalis as shown by A. Yaseen et al. [30].The authors observed that in the group with periodontitis, the prevalence was reported to be 88.9%, in the gingivitis group it was 84.9%, and in the healthy group it was 47.9%.Yaseen et al. [30].The authors observed that in the group with periodontitis, the prevalence was reported to be 88.9%, in the gingivitis group it was 84.9%, and in the healthy group it was 47.9%.

Results Based on the Gender
The prevalence of E. gingivalis in female patients was found to be 43%, lower than the prevalence in males, which was found to be 47%.In total, 14 out of 18 studies showed the prevalence difference between the genders is low.

Results Based on the Gender
The prevalence of E. gingivalis in female patients was found to be 43%, lower than the prevalence in males, which was found to be 47%.In total, 14 out of 18 studies showed the prevalence difference between the genders is low.

Results Based on Age
Only 8 of the 18 studies investigated specified a mean age for their sample size.The results showed that the increase in prevalence of E. gingivalis was correlated with an increase in the age of the patients.Study groups ranging from 25 to 45 years old and 45 to 60 years old revealed similar results.Two studies [27,28] reported a high prevalence of over 80% in the 45-to 60-year-old study group.The results were similar to what was reported by J. Luszcak et al. [16] in their study, recording the highest prevalence in the age groups of 40 to 49 and 50 to 59 years old.These results suggest that the prevalence of E. gingivalis is correlated with the age of the patients (Table 3).

Results Based on the Method of Detection Used
Most of the studies were conducted using the wet mount microscopy method, the collected sample being directly visualized under the microscope.Other methods included the DNA detection method (Garcia et [11,18,21,26,[28][29][30]).They revealed a prevalence of 53% of E. gingivalis.When using the wet mount microscopy method, studies revealed a prevalence of 40%.The direct wet mount microscopy method which counts the specific movement of E. gingivalis and its specific nucleus showed a prevalence of 40%, similar to the global prevalence of 43% (Figure 6).collected their samples from subgingival curettage.The results showed that samples from subgingival plaque had a mean prevalence of E. gingivalis of 56%, dental plaque and saliva samples of 35%, while samples taken using subgingival curettage showed 76% prevalence (Figures 8 and 9).subgingival plaque had a mean prevalence of E. gingivalis of 56%, dental plaque and saliva samples of 35%, while samples taken using subgingival curettage showed 76% prevalence (Figures 8 and 9).

Discussion
In this study, the authors looked into the prevalence of E. gingivalis in periodontal disease.A substantial risk to the public's health is posed by the high incidence of periodontal disease (gingivitis and periodontitis) among people of all ages, which can lead to tooth loss [31][32][33][34].Some key factors of periodontitis have not been identified yet [5].In the

Discussion
In this study, the authors looked into the prevalence of E. gingivalis in periodontal disease.A substantial risk to the public's health is posed by the high incidence of periodontal disease (gingivitis and periodontitis) among people of all ages, which can lead to tooth loss [31][32][33][34].Some key factors of periodontitis have not been identified yet [5].In the etiology of periodontal diseases, the bacterial factor has been described numerous times [4,35,36].On the other hand, parasites have not been investigated throughout.With the new advancement in the scientific world that proved that E. gingivalis is causing tissue damage, [11] our study aimed to evaluate the prevalence of these species in correlation with periodontal tissues.The prevalence of E. gingivalis reported in the case-control groups, which were not diagnosed with oral disease, was 18%.It was lower than in the periodontal disease group.These results are similar to those reported by other authors like Badri et al. [37] and X. Bao et al. [11].They showed that the prevalence of E. gingivalis was also significantly higher in periodontal disease cases compared to the case-control study group.The increased prevalence in some of the case-control groups can result from different methods used or smaller sample sizes.Interestingly to note, studies that showed a higher prevalence in the case-control group used the wet mount microscopy method.The results showed that the prevalence of E. gingivalis in patients with gingivitis, which is characterized by no attachment loss, was higher than in patients with periodontitis, characterized by a certain degree of attachment loss.This is contrary to other authors that described that the prevalence increases in periodontal disease severity [30,37,38].The results could be explained by a different study selection and the different methods used by the studies investigated.The results of the methods used also showed a discrepancy between the DNA detection and the other methods (microscopic approach).The DNA detection method showed a prevalence of E. gingivalis of 53% while the wet mount microscopy method showed only a prevalence of 40%.Compared to different staining methods, this discrepancy becomes even larger (Gomori's modified trichrome staining at 17%, Giemsa staining at 35%, and iron hematoxylin staining at 29%).These discrepancies come probably from the subjectivity of the microscopic approach, which depends on the examiner's knowledge and experience, the number of fields examined, the type of microscope used (light versus phase contrast), the nature of mounting media, and the delay between sampling and examination.The last factor is especially crucial for the wet mount microscopy method because the mobility of E. gingivalis is the identification factor of the parasite, as described by Bonner et al. [13].Prevalence based on gender was 43% in female patients and 47% in male patients, not a significant difference.These results are in accordance with previous researchers [21,24,26].When comparing the different sample methods, it is clear that the subgingival plaque sample results in higher prevalence compared to the dental plaque/saliva sampling method.This can be an indicator that E. gingivalis is most found in periodontitis when compared to gingivitis.The finding was also described by Bonner et al. in their previous research [13].
Further studies, like the very comprehensive meta-analysis performed by Badri et al. (2022) [37], concluded that a high prevalence of E. gingivalis of 77% was found among periodontal disease patients.Furthermore, Bonner et al. [39] demonstrated that infection by E. gingivalis and periodontitis are correlated and E. gingivalis is a very common parasite among humans.
Martin-Garcia et al. (2022) [40] examined the prevalence of these parasites in the periodontal pockets and concluded that E. gingivalis was more abundant in periodontal pockets that in healthy sulcus with a general prevalence of 76.9% (95% CI).Further studies, like Trim et al. [41], studied the prevalence of E. gingivalis using DNA detection based on a real-time PCR assay and concluded that this method could measure parasite loads and determine if treatments are efficacious in elimination of these parasites.Jiao et al. (2022) [42] conducted an rRNA gene sequencing study to detect both E. gingivalis and bacterial microbiome and concluded that 60% of the research participants (n = 60) harbored E. gingivalis in the subgingival plaque.Further studies, like Dubar et al. [21] showed that E. gingivalis was found to be significantly higher in pathological regions compared to healthy regions of control or periodontitis patients.
Future research examination should also take the underestimated prevalence in dental plaque and saliva into account.It should be considered in any future research attempting to link oral parasites in health and disease especially given the availability of an experimentally validated protocol for such an intention.The application of quantitative PCR could have resolved the association between the parasite load and periodontal disease.The strain diversity of E. gingivalis, which has two main variations, should be considered in future research, and a larger sample size is required to determine the biological significance of these E. gingivalis subtypes.Future studies should also take into account other studies published in different languages.

Conclusions
Our analysis concludes that there is an increased prevalence of entamoebas in oral samples from people with periodontal diseases compared to periodontally healthy subjects.Further research should ensure a clear selection of the samples given that the direct/microscopic approach can result in an underestimation of the prevalence of E. gingivalis.

Figure 2 .
Figure 2. Mean values of the prevalence of Entamoeba gingivalis.

Figure 2 .
Figure 2. Mean values of the prevalence of Entamoeba gingivalis.

Figure 3 .
Figure 3. Mean values of the prevalence of Entamoeba gingivalis associated with gingivitis.

Figure 3 .
Figure 3. Mean values of the prevalence of Entamoeba gingivalis associated with gingivitis.

Figure 3 .
Figure 3. Mean values of the prevalence of Entamoeba gingivalis associated with gingivitis.

Figure 4 .
Figure 4. Mean values of the prevalence of E. gingivalis associated with periodontitis.

Figure 4 .
Figure 4. Mean values of the prevalence of E. gingivalis associated with periodontitis.

Figure 5 .
Figure 5. Comparative results of the prevalence of E. gingivalis in the study groups vs control groups.

Figure 5 .
Figure 5. Comparative results of the prevalence of E. gingivalis in the study groups vs control groups.

Figure 6 .
Figure 6.Number of infected patients based on the method used for analysis.

Figure 7 .
Figure 7. Prevalence based on the staining method used.

AFigure 7 .
Figure 7. Prevalence based on the staining method used.

3. 6 .
Results Based on Location of the Samples A total of 16 out of the 18 studies investigated specified the location where the sample was taken from.J. Luszak et al., El.Dardiry et al., G. Garcia et al., M. Dubar et al., O. Arpag et al., and B.N. Al-Nuaimi et al. collected their sample from subgingival plaque by using sterile swabs in subgingival areas.The other nine authors, except X. Bao et al., collected their samples from saliva and dental plaque with the help of a sterile swab.X. Bao et al.

Figure 8 .
Figure 8. Prevalence based on the sample used (Dental plaque/saliva).

Figure 8 .
Figure 8. Prevalence based on the sample used (Dental plaque/saliva).

Figure 8 .
Figure 8. Prevalence based on the sample used (Dental plaque/saliva).

Table 1 .
Inclusion and exclusion criteria within the study framework.

Table 2 .
Main characteristics of the studies examined in the research.

Table 3 .
Prevalence highly correlated with the age of the patients.

Based on the Age of the Research Participants
al., M. Dubar et al., X. Bao et al., Al-Jubory et al., Al-Sarhan et al., Stensvold et al., and A. Yaseen et al.